title: Measurement of cationic and intracellular modulation of integrin binding affinity by AFM-based nanorobot.
abstract: Integrins are dynamic transmembrane cation-dependent heterodimers that both anchor cells in position and transduce signals into and out of cells. We used an atomic force microscope (AFM)-based nanorobotic system to measure integrin-binding forces in intact human intestinal epithelial Caco-2 cells. The AFM-based nanorobot enables human-directed, high-accuracy probe positioning and site-specific investigations. Functionalizing the AFM probe with an arginine-glycine-aspartate (RGD)-containing sequence (consensus binding sequence for integrins) allowed us to detect a series of peptide-cell membrane interactions with a median binding force of 115.1 ± 4.9 pN that were not detected in control interactions. Chelating divalent cations from the culture medium abolished these interactions, as did inhibiting intracellular focal adhesion kinase (FAK) using Y15. Adding 1 mM Mg(2+) to the medium caused a rightward shift in the force-binding curve. Adding 1 mM Ca(2+) virtually abolished the RGD-membrane specific interactions and blocked the Mg(2+) effects. Cell adhesion assays demonstrated parallel effects of divalent cations and the FAK inhibitor on cell adhesion. These results demonstrate direct modulation of integrin-binding affinity by both divalent cations and intracellular signal inhibition. Additionally, three binding states (nonspecific, specific inactivated, and specific activated) were delineated from affinity measurements. Although other research has assumed that this process of integrin conformational change causes altered ligand binding, in this work we directly measured these three states in individual integrins in a physiologically based study.
authors.affiliation: College of Human Medicine, Michigan State University, East Lansing, Michigan, USA.
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Patterson
authors.forename: Ruiguo
authors.initials: R
authors.lastname: Yang
authors.forename: Bixi
authors.initials: B
authors.lastname: Zeng
authors.forename: Bo
authors.initials: B
authors.lastname: Song
authors.forename: Shouye
authors.initials: S
authors.lastname: Wang
authors.forename: Ning
authors.initials: N
authors.lastname: Xi
authors.forename: Marc D
authors.initials: MD
authors.lastname: Basson
mesh_terms: D018938:Caco-2 Cells; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D042541:Intracellular Space / Q000201:enzymology / Q000378:metabolism*; D018625:Microscopy, Atomic Force / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding; D012371:Robotics
chemical_list: D016023:Integrins; D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid